This application addresses broad Challenge Area (14) Stem Cells, and specific Challenge Topic 14-DK-104: In vitro differentiation of human Embryonic Stem Cells (ES)/Induced Pluripotent Stem Cells (iPS) to NIDDK relevant cells/tissues. Hemoglobinapathies, such as sickle cell disease and [unreadable]-thalassemia, are anemias of varying intensity that continue to have a deleterious impact on the health of specific subpopulations in the US workforce, ultimately exerting a negative effect on our economic productivity. We have an interest in developing novel approaches to alter the normal hemoglobin switching mechanism so that patients with hemoglobinapathies might benefit from derepression of fetal or embryonic globin expression. Briefly, the idea described in the present proposal involves using human embryonic stem (ES) cells, engineered to express detectable reporters from their fetal and adult globin promoters, to screen for small molecules that lead to more efficient induction of adult globin, or reactivation of fetal globin, expression. The major advantages of this approach is that it avoids the historical use of the mouse to address what is a human gene regulatory pattern, and that it takes advantage of the normal hematopoietic process observed during ES cell differentiation, a process that cannot be recapitulated within human leukemic cell lines. As a result, this application proposes to define and utilize a novel approach to alter the normal hemoglobin switching mechanism via three interrelated aims: 1) establish efficient hES cell differentiation conditions that correctly recapitulate the normal [unreadable]-like globin gene developmental sequence;2) generate a correctly regulated reporter hES cell line that mimics this endogenous expression pattern;3) use this line to screen a small molecule library for chemicals that alter/redirect [unreadable]-like globin expression. A key aspect of this design is that the chemical screen in Aim 3, and thus the ultimate success of the proposal, is not solely dependent on establishment of adult [unreadable]-producing cells in Aim 1. This fits well into the 14-DK-104 category, as it aims to identify small molecules that redirect hemoglobin expression in differentiating hES cells and is consistent with programmatic interests of the NIDDK. PUBLIC HEALTH RELEVANCE: The goals of this project are to establish a molecular and cellular baseline of marked [unreadable].like globin gene switching in human embryonic stem cells that will then provide a rigorous and directly relevant avenue for testing inducers of adult globin or reactivators of fetal globin expression.